The alpha2-adrenergic receptors (ARs) belong to the G-protein coupled receptor (GPCR) super-family and mediate a number of physiological/pharmacological responses, which can lead to both favorable and adverse effects when the alpha2-AR is exploited as a therapeutic target for treatment of diseases such as ADHD. The long-term goal of our studies is to understand the mechanisms by which alpha-2AR functions are regulated at the molecular and cellular levels, so as to provide new insights for therapeutic strategies. The properties of different agonists in stimulating various alpha2-AR-mediated responses and in inducing alpha2-AR internalization and subsequent sorting in native cells are certainly the bases for alpha2-AR physiological functions. However, to date, a clear understanding of these processes remains elusive. As a unique tool, I have generated a mouse line in which N-terminal epitope-tagged HA-alpha2A-AR expression is driven by the endogenous mouse alpha2A-AR locus (HA-alpha2A-AR knock-in). In this study, we will utilize this novel line to evaluate regulation of alpha2A-AR trafficking and signaling by two agonists, clonidine and guanfacine, in comparison with the endogenous ligand, norepinephrine, in native neocortical neurons. Although both agonists are used to treat ADHD and can improve cognition through activation of the alpha2A-AR, clonidine and guanfacine exhibit differential capacities to elicit sedation, an undesirable side-effect. Our hypothesis is that the difference between these two agonists in eliciting in vivo responses is due to their diverse capabilities in inducing alpha2A-AR trafficking and signaling responses in native neurons. Two Specific Aims are proposed to test this hypothesis: 1) We will label surface HA-alpha2A-AR in live neocortical cultures derived from the HA-alpha2A-AR knock-in mice for fluorescent and colorimetric detection, and determine the potency and efficacy of each agonist in inducing receptor internalization as well as receptor postendocytotic sorting profiles; 2) We will evaluate the efficacy and potency of each agonist in stimulating two signaling pathways evoked by the alpha2A-AR; inhibition of voltage-gated calcium current and activation of MAP kinase in neocortical neurons. Given the fact that one kind of GPCR often regulates multiple physiological responses, identification and development of response-specific agonists represents a novel and promising therapeutic strategy for treating a variety of disease states while restricting undesirable side effects. Revealing the molecular and cellular basis of alpha2-AR regulation by different agonists will be been important for guiding the development and subsequent utilization of alpha2-AR agonists in ADHD treatment as well as in other clinical settings where use of alpha2AR-agonists may be warranted. [unreadable] [unreadable] [unreadable]